This proposal focuses on the development of a highly sensitive and specific multiplex immune marker quantification tool for monitoring host immunoglobulin responses against Lyme and other blacklegged tick-borne pathogens using Antibody Detection by Agglutionation-PCR (ADAP) technology. Lyme disease (LD) is often missed or unrecognized and if not treated definitively during early infection, patients can suffer severe and often irreversible consequences. In the US, infection with the Lyme disease pathogen (Borrelia burgdorferi) is increasingly prevalent, affecting over 300,000/yr. Unlike other bacterial infections, direct detection of LD bacterial genomic materials and antigens is not useful in early diagnosis due to the extreme scarcity of LD bacteria in the circulation. The two-tier serological testing algorithm (first-tier enzyme immunoassay followed by a second-tier Western blot) is still the mainstay of Lyme testing. However, this two-tier algorithm identifies less than 40% of early LD infections, signaling an urgent and compelling need for a better diagnostic assay. In addition, ticks transmitting LD can also contain other pathogens causing debilitating co-infections requiring accurate differential diagnosis. In particular, (Borrelia miyamotoi-induced) Relapsing Fever and Babesiosis are two common diseases requiring different antimicrobial management. This project proposes to adapt and employ our novel ADAP technology that harnesses the exponential amplification attributes of PCR to achieve highly sensitive detection of host antibody immune responses. ADAP's DNA barcoding will be used to enable simultaneous multiplex quantification of immune markers against several Lyme, Relapsing Fever and Babesiosis antigens in 1-2 ?L of samples. The proposed multiplex ADAP tick-borne disease assay's enhanced sensitivity can enable much earlier disease detection during the critical window period for best outcomes and reveal possible co-infection/s for most appropriate treatment. In Specific Aim 1, we will express and purify several Lyme recombinant proteins that are not currently commercially available. Then, we will synthesize corresponding antigen-DNA conjugate pairs for a ADAP Lyme assay. Finally, we will optimize and validate the assay's performance using well annotated patient samples from our Co-Investigator's sites at Columbia University in New York, Tulane in New Orleans and from the CDC. Notably, we present a plan to rigorously investigate and mitigate potential cross-reactivity with other diseases, such as syphilis and rheumatoid arthritis. In Specific Aim 2, we will further express and purify antigens for Relapsing Fever and Babesiosis and then synthesize and validate corresponding antigen-DNA conjugates using patient samples. If successful, conjugates from Aim 1 and 2 will be combined into a single multiplex tick-borne disease assay for blacklegged tick-transmitted pathogens. The positive outcome of this project will be a comprehensive assay able to identify patients during the earliest, most valuable treatment window, offering an unprecedented opportunity to substantially improve outcomes and reduce the profound personal and societal burden of chronic disease.